Riboflavin (vitamin B2) is synthesized by all plants and many microorganisms but is not produced by higher animals. Because it is a precursor to coenzymes such as flavin adenine dinucleotide and flavin mononucleotide that are required in the enzymatic oxidation of carbohydrates, riboflavin is essential to basic metabolism. In higher animals, insufficient riboflavin can cause loss of hair, inflammation of the skin, vision deterioration, and growth failure.
The enzymes required catalyzing the biosynthesis of riboflavin from guanosine triphosphate (GTP) and ribulose-5-phosphate are encoded by four genes (ribG, ribB, ribA, and ribH) in B. subtilis. These genes are located in an operon, the gene order of which differs from the order of the enzymatic reactions catalyzed by the enzymes. For example, GTP cyclohydrolase II, which catalyzes the first step in riboflavin biosynthesis, is encoded by the third gene in the operon, ribA. The ribA gene also encodes a second enzymatic activity, i.e., 3,4-dihydroxy-2-butanone 4-phosphate synthase (DHBPS), which catalyzes the conversion of ribulose-5-phosphate to the four-carbon unit 3,4-dihydroxy-2-butanone 4-phosphate (DHBP). Deaminase and reductase are encoded by the first gene of the operon, ribG. The penultimate step in riboflavin biosynthesis is catalyzed by lumazine synthase, the product of the last rib gene, ribH. Riboflavin synthase, which controls the last step of the pathway, is encoded by the second gene of the operon, ribB. The function of the gene located at the 3′ end of the rib operon is, at present, unclear; however, its gene product is not required for riboflavin synthesis.
Transcription of the riboflavin operon from the ribP1 promoter is controlled by an attenuation mechanism involving a regulatory leader region located between ribP1 and ribG. ribO mutations within this leader region result in deregulated expression of the riboflavin operon. Deregulated expression is also observed in strains containing missense mutations in the ribC gene. The ribC gene has been shown to encode the flavin kinase/FAD synthase of B. subtilis (Mack, M., et al., J. Bacteriol., 180:950-955, 1998). Deregulating mutations reduce the flavokinase activity of the ribC gene product resulting in reduced intracellular concentrations of flavin mononucleotide (FMN), the effector molecule of the riboflavin regulatory system.
Engineering of riboflavin production strains with increased production rates and yields of riboflavin has been achieved in the past in a number of different ways. For instance, (1) classical mutagenesis was used to generate variants with random mutations in the genome of the organism of choice, followed by selection for higher resistance to purine analogs and/or by screening for increased production of riboflavin. (2) Alternatively, the terminal enzymes of riboflavin biosynthesis, i.e., the enzymes catalyzing the conversion of guanosine triphosphate (GTP) and ribulose-5-phosphate to riboflavin, were over-expressed, resulting also in a higher flux towards the target product. The metabolic flux into and through a biosynthetic pathway, e.g. the riboflavin biosynthetic pathway, is determined by the specific activities of the rate-limiting enzymes of this particular pathway and by the intracellular concentrations of the substrates for these enzymes. Only at or above saturating substrate concentrations an enzyme can operate at its maximal activity. The saturating substrate concentration is a characteristic feature for each enzyme. For example, the metabolic flux into the riboflavin pathway may be increased or kept at a high level by keeping the intracellular concentrations of ribulose-5-phosphate above or as close as possible to the saturating substrate concentration of the 3,4-dihydroxy-2-butanone 4-phosphate synthase, a presumed rate limiting enzyme for the riboflavin biosynthetic pathway. High intracellular concentrations of ribulose-5-phosphate may, for example, be reached by preventing or interfering with drainage of ribulose-5-phosphate into the central metabolism via the non-oxidative part of the pentose phosphate pathway.
A key enzyme in the non-oxidative part of the pentose phosphate pathway is the transketolase enzyme, which catalyzes the reversible conversion of ribose-5-phosphate and xylulose-5-phosphate to seduheptulose-7-phosphate and glyceraldehyde-3-phosphate. In addition, transketolase catalyzes also the conversion of fructose-6-phosphate and glyceraldehyde-3-phosphate to xylulose-5-phosphate and erythrose-4-phosphate (Kochetov, G. A. 1982, Transketolase from yeast, rat liver, and pig liver, Methods Enzymol., 90:209-23).
It has previously been reported that transketolase deficient Bacillus subtilis strains carrying knock-out mutations in the transketolase encoding gene produces ribose, which accumulates in the fermentation broth (De Wulf, P., and E. J. Vandamme. 1997. Production of D-ribose by fermentation, Appl. Microbiol. Biotechnol. 48:141-148; Sasajima, K., and Yoneda, M. 1984, Production of pentoses by microorganisms. Biotechnol. and Genet. Eng. Rev. 2: 175-213). Obviously, increased intracellular C5 carbon sugar pools can be reached in transketolase knock-out mutants up to a level that exceeds the physiological requirements of the bacteria and leads to secretion of excess ribose.
As mentioned above, transketolase catalysed reactions are also required to produce erythrose-4-phosphate, from which the three proteinogenic aromatic amino acids are derived. Therefore, transketolase deficient microorganisms are auxotroph for these amino acids. They can only grow if these amino acids or their biosynthetic precursors, for instance shikimic acid, can be supplied via the cultivation medium.
In addition to the unfavorable auxotrophy for aromatic amino acids or shikimic acid, transketolase-deficient B. subtilis mutants show a number of severe pleiotropic effects like very slow growth on glucose, a defective phosphoenolpyruvate-dependent phosphotransferase system, deregulated carbon catabolite repression, and altered cell membrane and cell wall composition (De Wulf, P., and E. J. Vandamme. 1997).
An other transketolase-deficient riboflavin secreting B. subtilis strain was described by Gershanovich et al. (Gershanovich V N, Kukanova A I a, Galushkina Z M, Stepanov A I (2000) Mol. Gen. Mikrobiol. Virusol. 3:3-7).
Furthermore, U.S. Pat. No. 6,258,554 B1 discloses a riboflavin overproducing Corynebacterium glutamicum strain in which transketolase activity is deficient. It can be noted form the disclosure of the U.S. Pat. No. 6,258,554 B1 that the deficiency in transketolase activity and the resulting amino acid auxotrophy was essential for the improved riboflavin productivity, since a prototrophic revertant produced riboflavin in amounts similar to a C. glutamicum strain with a wild-type transketolase background.
These disadvantages, i.e. auxotrophy for aromatic amino acids and further pleiotropic effects discussed above, make a transketolase deficient mutant a less preferable production strain for stable industrial processes, such as, e.g. the industrial production of riboflavin within such strain.